Amino-acid Sequence of λ Phage Endolysin

WE wish to present a preliminary report of the amino-acid sequence of λ endolysin. This protein is a lytic enzyme¹ and its structural gene, R, maps toward the right end of λ DNA². Conditional mutants as well as frame-shift mutants (R. Thomas, personal communication) have been isolated and analysed³. Hogness et al.⁴ developed a technique to assay the gene activity of the fragmented λ DNA, which suggested that it might be possible to isolate a small segment of DNA containing the endolysin gene. Purification, immunological properties and end group analysis of λ endolysin were studied by Black and Hogness^5–7.     

Sequence preference of α-helix N-terminal tetrapeptide

The α-helix is the most abundant secondary structure in proteins. Due to the specific i, i+4 hydrogen bond pattern, the two termini have unsatisfied hydrogen bonds, and are less constrained; in order to compensate for this, specific residues are preferred for the terminal positions. However, a naive combination of the statistically-preferred residues for each position may not result in a stable N-terminal helical sequence. In order to provide a set of preferable N-terminal peptides for α-helix design, we have studied the N-terminal tetrapeptide sequence motifs that are favorable for helix formation using statistical analysis and atomistic simulations. A set of tetrapeptide sequences including TEEE and TPEE were found to be favorable motifs. In addition to forming more hydrogen bonds in the helical conformation, the favorable motifs also tended to form more capping boxes. To empirically test our predictions, we obtained 10 peptides with different N-terminal motifs and measured their α-helical content by circular dichroism spectroscopy. The experimental results agreed qualitatively with the statistical and simulation results. Furthermore, some of the suggested preferable tetrapeptide sequences have been successfully applied in de novo protein design.     

Sequence analysis of sheep T-cell receptor β chains

The nucleotide sequences reported in this paper have been submitted to the GenBank database and have been assigned the accession numbers M94181-M94183.     

Complete Genome Sequence of a Pantón-Valentine Leukocidin-Negative Community-Associated Methicillin-Resistant Staphylococcus aureus Strain of Sequence type 72 from Korea

In the past decade, community-associated (CA-) infections with methicillin-resistant Staphylococcus aureus (MRSA) have emerged throughout the world. Different CA-MRSA strains dominate in different geographical locations. Many CA-MRSA lineages contain genes coding for the Pantón-Valentine leukocidin. However, the role of this leukotoxin in CA-MRSA pathogenesis is still controversial. The genome sequences of two key PVL-positive CA-MRSA strains (USA300, USA400) have been reported, but we lack information on the more recently found PVL-negative CA-MRSA strains. One such strain is the PVL-negative ST72, the main cause of CA-MRSA infections in Korea. Here, we report the entire genome sequence of CA-MRSA ST72 and analyze its gene content with a focus on virulence factors. Our results show that this strain does not have considerable differences in virulence factor content compared to other CA-MRSA strains (USA300, USA400), indicating that other toxins do not substitute for the lack of PVL in ST72. This finding is in accordance with the notion that differential expression of widespread virulence determinants, rather than the acquisition of additional virulence factors on mobile genetic elements, such as PVL, is responsible for the increased virulence of CA- compared to hospital-associated MRSA.     

Can You Sequence Ecology? Metagenomics of Adaptive Diversification

Few areas of science have benefited more from the expansion in sequencing capability than the study of microbial communities. Can sequence data, besides providing hypotheses of the functions the members possess, detect the evolutionary and ecological processes that are occurring? For example, can we determine if a species is adapting to one niche, or if it is diversifying into multiple specialists that inhabit distinct niches? Fortunately, adaptation of populations in the laboratory can serve as a model to test our ability to make such inferences about evolution and ecology from sequencing. Even adaptation to a single niche can give rise to complex temporal dynamics due to the transient presence of multiple competing lineages. If there are multiple niches, this complexity is augmented by segmentation of the population into multiple specialists that can each continue to evolve within their own niche. For a known example of parallel diversification that occurred in the laboratory, sequencing data gave surprisingly few obvious, unambiguous signs of the ecological complexity present. Whereas experimental systems are open to direct experimentation to test hypotheses of selection or ecological interaction, the difficulty in “seeing ecology” from sequencing for even such a simple system suggests translation to communities like the human microbiome will be quite challenging. This will require both improved empirical methods to enhance the depth and time resolution for the relevant polymorphisms and novel statistical approaches to rigorously examine time-series data for signs of various evolutionary and ecological phenomena within and between species.     

Complete Genomic Sequence of Transmissible Gastroenteritis Virus TS and 3＇ End Sequence Characterization Following Cell Culture

The complete genome sequence of transmissible Gastroenteritis virus (TGEV) strain TS, previously isolated from Gansu province, was cloned and compared with published sequence data from other TGEV strains.Phylogenetic tree analysis based on the amino acid and nucleotide sequences of the S gene showed that the TGEV strains were divided into 3 clusters. TGEV TS showed a close evolutionary relationship to the American Miller cluster but had a 5' non-translated region (NTR) sequence closely related to the American Purdue cluster.Continued culture in different cell types indicated that TGEV TS virulence could be attenuated alter fifty passages in Porcine kidney (PK-15) cells, and that the Porcine kidney cell line IB-RS-2 (IBRS) was not suitable for culture of the TGEV strain TS.     

Amino Acid Sequence of αkSubstance,a Mating Pheromone of Saccharomyces kluyveri

A fraction containing IgA (IgA-rich fraction) was prepared from bovine colostrum by anion exchange chromatography using DEAE-Sephadex A-50 and gel filtration on Sephadex G-200. A large amount of IgG1-dimer was found in this fraction, which could not be separated from IgA by repeated gel filtration.

The Fc fragment of bovine colostral IgG (IgG-Fc) was prepared from papain digestion mixtures. IgG-Fc was found to be heterogeneous on DEAE-cellulose column chromatography. Two IgG-Fc fractions were obtained, but no antigenic difference was found between them. Anti-IgG-Fc antibodies raised in rabbits by injection of these Fc preparations reacted only with IgG1 and IgG2. An immunoadsorbent (anti-IgG-Fc-Sepharose) was prepared by coupling these anti-IgG-Fc antibodies to CNBr-activated Sepharose 4B.

IgA was purified from the IgA-rich fraction by affinity chromatography on anti-IgG-Fc-Sepharose adsorbent. IgG1-dimer was effectively removed by this treatment. The purified sample gave only one precipitin arc characteristic of IgA on immunoelectrophoresis with multiple anti-bovine colostral whey antiserum. A small amount of IgA was found to be adsorbed to the affinity column nonspecifically.

When a rabbit was immunized with the purified IgA, besides anti-IgA antibodies, antibodies against the secretory component (SC) were found in the antiserum. This finding leads us to expect that the purified IgA is secretory IgA containing SC.     

Oligonucleotide Sequence of Replicase Initiation Site in Qβ RNA

THE single stranded RNA genome of bacteriophage Qβ has been variously estimated to consist of from 3,500¹ to 4,500² nucleotides. It contains three known cistrons³, which correspond to three of the four Qβ-specific proteins synthesized in vivo and in vitro^4–6. These are: (1) the gene for the maturation or A protein (molecular weight 41,000 (refs. 4, 5)), (2) that for the major coat protein of the virus (molecular weight 14,000 (ref. 9)) and (3) the gene for the phage-specific subunit of the Qβ replicase (molecular weight 64,000 (ref. 10) or 69,000 (ref. 24)), listed in the probable order^7,8 that they occur on the Qβ RNA. The fourth Qβ-specific protein, A₁ or IIb (molecular weight 36,000 (refs. 4–6, 10)), has recently been shown by Weiner and Weber to have an N-terminal sequence which is identical (for eight amino-acids) to that of the coat protein⁷. Because increased amounts of A₁ appear in virus particles grown in cells containing a UGA suppressor, Weiner and Weber postulate⁷ that this protein is the product of natural read-through at the UGA termination signal of the Qβ coat cistron. Such read-through (involving about 600 nucleotides) could occur entirely within a large “intercistronic” region between the coat and replicase genes, or could involve translation, either in or out of phase, of the replicase cistron. In hopes of distinguishing between these alternatives, I have isolated and examined the nucleotide sequence of the region surrounding the initiator codon of the Qβ replicase gene.     

Complete Genome Sequence of Serratia plymuthica Bacteriophage ?MAM1

A virulent bacteriophage (ϕMAM1) that infects Serratia plymuthica was isolated from the natural environment and characterized. Genomic sequence analysis revealed a circular double-stranded DNA sequence of 157,834 bp, encoding 198 proteins and 3 tRNAs. The ϕMAM1 genome shows high homology to previously reported ViI-like enterobacterial bacteriophage genomes.     

Sequence and diversity of bovine T-cell receptor β-chain genes

The nucleotide sequences of 38 T-cell receptor (Tcr) -chain cDNA clones which were isolated from a cDNA library (2 × 10⁶ plaques) constructed from bovine peripheral blood lymphocytes were determined. Of 38 cDNA clones, 22 were rearranged and contained the functional variable (V) gene segments. These clones were tentatively divided into nine Tcrb-V gene families which correspond to the human Tcrb-V family. Among them, a Tcrb-V12 gene segment was isolated from 9 out of 22 clones, suggesting that this Tcrb-V family was expressed in the bovine peripheral blood lymphocytes. Two different constant (C) geen segments were found, and both C regions were composed of 178 amino residues. The amino acid sequences of bovine Tcrb-C regions are approximately 80%–82%, 78%, and 78% similar to those from human, mouse, and rabbit, respectively. To estimate Tcrb-V-associated restriction fragment length polymorphisms (RFLPs), Southern blot analysis was performed using liver DNAs from four bovine breeds, Holstein, Angus, Hereford, and Japanese Black. However, no significant difference was observed among genomic DNAs of Tcrb-V loci from these four breeds.The nucleotide sequence data reported in this paper have been submitted to the GenBank nucleotide sequence database and have been assigned the accession numbers D90121-40. Address correspondence and offprint requests to: N. Ishiguro.     

Isolation and Sequence Analysis of Bovine αsl-Casein cDNA Clone

Nucleoside oxidase, a novel nucleoside oxidizing enzyme has been purified from a crude extract of Pseudomonas maltophilia LB-86 by a ammonium sulfate fractionation, heat treatment, column chromatography on DEAE-Toyopearl, and gel filtration twice on Sephacryl S-200. The overall purification was approximately 60-fold with a yield of 35 %. The purified enzyme gave a single protein band on acrylamide gel electrophoresis.

Reaction products from inosine were identified as inosine-5′-aldehyde and inosine-5′-carboxylic acid. The enzyme catalyzed the oxidation of inosine to inosine-5′-carboxylic acid via inosine-5′-aldehyde using molecular oxygen as a primary electron acceptor with no formation of hydrogen peroxide.     

Sequence analysis of linked maize 22 kDa α-zein genes

We have determined the nucleotide sequence of a 7343 bp zein genomic clone (gZ22.8H3) from the maize inbred W64A. Computer-aided analysis of the DNA sequence revealed two contiguous 22 kDa -zein genes. The 5 gene (gZ22.8) encodes a complete polypeptide and contains putative regulatory sequences in both the 5 and 3 flanking regions that are typical of zein genes. In contrast, the 3 gene (gZ22.8) appears to be a pseudogene, because it contains numerous insertions and deletions that would prevent translation of the mRNA. Alignment of the 5 and 3 flanking sequences of both genes indicated that they resulted from a 3.3 kb DNA duplication event.     

The 5′ Nucleotide Sequence of Vesicular Stomatitis Viral RNA

The 5' nucleotide sequence of vesicular stomatitis viral RNA is pppApCpGp....     

Nucleotide Sequence of α-Galactosidase MEL Gene from Zygosaccharomyces mrakii

The region encompassing the α-galactosidase MELr gene was amplified from Zygosaccharomyces mrakii IFO 1835^T by inverse-PCR and then sequenced. The nucleotide sequence of this region revealed a single open reading frame of 1410 bp encoding a 470 amino acid protein with a molecular weight of 51,909. The similarity of the deduced mature protein to other yeast α-galactosidases was 63.3% to Zygosaccharomyces cidri, 71.5% to Torulaspora delbrueckii, and 70.7–73.9% to Saccharomyces species. The nucleotide and amino acid sequences are deposited in the DDBJ/EMBL/GenBank database under Accession Number AB030209. Received: 10 February 2000 / Accepted: 29 March 2000     

Complete Genome Sequence of φHSIC, a Pseudotemperate Marine Phage of Listonella pelagia

The genome for the marine pseudotemperate member of the Siphoviridae HSIC has been sequenced using a combination of linker amplification library construction, restriction digest library construction, and primer walking. HSIC enters into a pseudolysogenic relationship with its host, Listonella pelagia, characterized by sigmoidal growth curves producing >10⁹ cells/ml and >10¹¹ phage/ml. The genome (37,966 bp; G+C content, 44%) contained 47 putative open reading frames (ORFs), 17 of which had significant BLASTP hits in GenBank, including a β subunit of DNA polymerase III, a helicase, a helicase-like subunit of a resolvasome complex, a terminase, a tail tape measure protein, several phage-like structural proteins, and 1 ORF that may assist in host pathogenicity (an ADP ribosyltransferase). The genome was circularly permuted, with no physical ends detected by sequencing or restriction enzyme digestion analysis, and lacked a cos site. This evidence is consistent with a headful packaging mechanism similar to that of Salmonella phage P22 and Shigella phage Sf6. Because none of the phage-like ORFs were closely related to any existing phage sequences in GenBank (i.e., none more than 62% identical and most <25% identical at the amino acid level), HSIC is unique among phages that have been sequenced to date. These results further emphasize the need to sequence phages from the marine environment, perhaps the largest reservoir of untapped genetic information.     

Systematic Dissection of the Sequence Determinants of Gene 3’ End Mediated Expression Control

The 3’end genomic region encodes a wide range of regulatory process including mRNA stability, 3’ end processing and translation. Here, we systematically investigate the sequence determinants of 3’ end mediated expression control by measuring the effect of 13,000 designed 3’ end sequence variants on constitutive expression levels in yeast. By including a high resolution scanning mutagenesis of more than 200 native 3’ end sequences in this designed set, we found that most mutations had only a mild effect on expression, and that the vast majority (~90%) of strongly effecting mutations localized to a single positive TA-rich element, similar to a previously described 3’ end processing efficiency element, and resulted in up to ten-fold decrease in expression. Measurements of 3’ UTR lengths revealed that these mutations result in mRNAs with aberrantly long 3’UTRs, confirming the role for this element in 3’ end processing. Interestingly, we found that other sequence elements that were previously described in the literature to be part of the polyadenylation signal had a minor effect on expression. We further characterize the sequence specificities of the TA-rich element using additional synthetic 3’ end sequences and show that its activity is sensitive to single base pair mutations and strongly depends on the A/T content of the surrounding sequences. Finally, using a computational model, we show that the strength of this element in native 3’ end sequences can explain some of their measured expression variability (R = 0.41). Together, our results emphasize the importance of efficient 3’ end processing for endogenous protein levels and contribute to an improved understanding of the sequence elements involved in this process.